Antisense oligonucleotides are oligonucleotides or analogs, whose sequence is complementary to a predetermined segment of mRNA. Typically, sequences of the antisense oligonucleotides are chosen so as to be complementary to a critical sequence in a gene so that if the gene, or mRNA transcribed therefrom, is hybridized to the complementary antisense sequence, the gene cannot be expressed or is subjected to enzymatic degradation (see U.S. Pat. No. 6,291,438, incorporated herein by reference).
The use of antisense oligonucleotides has been proposed for a variety of infections as well as proliferative disorders. In view of their sequence specificity, antisense oligonucleotides should be ideal anti-infective agents (Zamecnik and Stephenson, 1978; Summerton, 1979). However, their successful application in therapeutics has been delayed by problems of efficacy and drug delivery. Native oligonucleotides are not biomembrane-permeable. If delivered into cells with the help of amphipathic cations or liposomes, they could still be hydrolyzed by endogenous nucleases before reaching the intended targets. In order to improve bioavailability, various types of chemically modified antisense oligonucleotides have been made by solid-state synthesis and tested with different degrees of success (Summerton and Weller, 1997; Wang et al., 1999; Nesterova and Cho-Chung, 2000; Stein, 1997). In this regard, phosphorothioate oligodeoxynucleotides (PS-oligo DNA) are considered to be more resistant to nuclease digestion than corresponding phosphodiester oligodeoxynucleotides. Further, 2′-O-methyloligoribonucleotide phosphorothioates (PS-2′-O-methyloligo RNA) are considered to be more resistant to nucleases than PS-oligo DNA and can form duplexes with RNA with higher affinity. In addition, while PS-oligo RNA/RNA duplexes are not a substrate for RNase H, PS-oligo DNA/RNA duplexes are.
Solid-state synthesis of chemically modified RNA is expensive and could also lead to stereochemical complications. For example, in a solid-state synthesized 21-nt phosphorothioate, each chiral P-atom can be in PS or PR configuration so that the product is really a mixture of 220 isomers. This stereochemical heterogeneity could give rise to non-sequence-specific toxicity.
One promising type of oligonucleotide platform is poly-2′-O-(2,4-dinitrophenyl)-oligoribonucleotide (poly-DNP-RNA) which can be synthesized by in vitro transcription with native rNTPs followed by a single step derivatization reaction. The product has no chiral P-atoms and hence is stereochemically homogeneous. It was found that poly-DNP-RNA with DNP/nucleotide molar ratio of 0.65 to 0.75 can rapidly and spontaneously cross viral envelopes (Ashun et al., 1996). It can also slowly and spontaneously cross mammalian cell membranes without transfection reagents (Ru et al., 1999). Poly-DNP-RNAs are also resistant to degradation by ribonucleases (Rahman et al., 1996; Wang, 1996).
Several antisense poly-DNP-RNAs have been synthesized and found to inhibit viral replication and cancer growth in a sequence-specific and concentration-dependent way with no non-sequence-specific toxicity in the effective concentration range (Xin and Wang, 1998; Ru et al., 1998; Ru et al., 1999). In situ hybridization experiments showed that after staying inside cancer cells for 72 h at 37° C., an antisense poly-DNP-RNA was structurally still sufficiently intact to hybridize with a biotin-labeled sense DNA probe (Ru et al., 1999). A successful in vivo application of poly-DNP-RNA has been reported in the treatment of murine leukemia. It was observed that either i.p. or oral administration of antisense poly-DNP-RNA to MMLV-infected mice eliminated not only viremia but also the DNA of the integrated viral genome in bone marrow (Wang and Wang, 1999). The observed elimination of integrated viral genome in bone marrow by oral administration of antisense poly-DNP-RNA highlighted the bioavailability of these agents. Apparently a sufficient number of the inhibitor molecules had passed through all the membrane barriers between the alimentary canal and bone marrow and reached their target in bone marrow to trigger the elimination of the infected cells, both active and resting. The infected mice that had not been treated, or those that were treated with poly-DNP-RNA of a wrong sequence all died, those that had been treated with antisense poly-DNP-RNA continued to live in apparent good health.
A common use of antisense oligonucleotides is in the field of cell proliferation disorders. An example of a gene considered to be important in cell growth regulation is the RIα/PKA gene. It is considered that RIα is an ontogenic growth-inducing protein, and its constitutive expression disrupts normal growth processes, resulting in proliferative disorders which can lead to malignancy. An increase in RIα/PKA is an early response to the mitogenic effects of growth factors, such as GM-CSF in human leukemic cells and TGF-α in normal rat fibroblast, and phytohemagglutinin stimulation of resting lymphocytes. Increased expression of RI has also been shown to be associated with both chemical and viral carcinogenesis and oncogene-induced cell transformation. RI is the major, or sole, R subunit of PKA detected in a variety of types of human cancer cell lines and primary tumors examined (Cho-Chung, 1997). The majority of human breast cancer and colon carcinomas examined show an enhanced expression of RI and a higher ratio of PKA-I/PKA-II as compared with normal counterparts (Miller et al., 1993; Bradbury et al., 1994). Importantly, the relative overexpression of the RIα subunit of PKA was associated with poor prognosis in patients with breast cancer. Conversely, downregulation of RIα by site-selective cAMP analogs produces growth arrest and differentiation in a wide variety of human and rodent cancer cell lines. In addition, retroviral vector-mediated overexpression of RIα provided direct evidence that RIα plays a role in cell proliferation by regulating cell cycle progression (Tortora et al., 1994). These studies provide evidence that RIα plays a critical role in cell proliferation. Therefore, RIα/PKA is an attractive target for therapeutic approaches to malignancy.
In one study, the poly-DNP-RNA which was antisense to the RIα subunit of the protein kinase A (RIα/PKA), was used to inhibit the growth of breast cancer cells which overexpressed this gene. The antisense poly-DNP RNA was found to be effective in a concentration dependent manner. Further, intraperitoneal administration of the antisense to SCID mice with transplanted MDA-MB-231 cells was found to inhibit the growth of the xenografts in concentration dependent manner to prevent metastasis and reduce mortality (Ru et al., 1999, Oncology Res., 11:505-512). This antisense oligonucleotide exhibited an IC50 of about 22 nM in MCF-7 cells. Accordingly, for the antisense oligonucleotides to be useful in the clinical setting, there continues to be a need to improve the efficacy of these compounds.